Hyeran Jang

The methylenetetrahydrofolate reductase C677T mutation induces cell-specific changes in genomic DNA methylation and uracil misincorporation: A possible molecular basis for the site-specific cancer risk modification

The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with a decreased risk of colon cancer although it may increase the risk of breast cancer. This polymorphism is associated with changes in intracellular folate cofactors, which may affect DNA methylation and synthesis via altered one‐carbon transfer reactions. We investigated the effect of this mutation on DNA methylation and uracil misincorporation and its interaction with exogenous folate in further modulating these biomarkers of one‐carbon transfer reactions in an in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA‐MB‐435 breast adenocarcinoma cells. In HCT116 cells, the MTHFR 677T mutation was associated with significantly increased genomic DNA methylation when folate supply was adequate or high; however, in the setting of folate insufficiency, this mutation was associated with significantly decreased genomic DNA methylation. In contrast, in MDA‐MB‐435 cells, the MTHFR 677T mutation was associated with significantly decreased genomic DNA methylation when folate supply was adequate or high and with no effect when folate supply was low. The MTHFR 677T mutation was associated with a nonsignificant trend toward decreased and increased uracil misincorporation in HCT116 and MDA‐MB‐435 cells, respectively. Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR 677T mutation in cells derived from the target organs of interest, thus providing a plausible cellular mechanism that may partly explain the site‐specific modification of colon and breast cancer risks associated with the MTHFR C677T mutation.

Genetic and Epigenetic Interactions between Folate and Aging in Carcinogenesis

Folate is among the most strongly implicated dietary components to convey protection against colon cancer, and diminished folate status is associated with an enhanced risk of colon cancer. Age is also regarded as one of the most important risk factors for colonic carcinogenesis. It is therefore of considerable interest to determine whether the process of aging influences folate metabolism in the colon and whether folate supplementation might prevent the procarcinogenic effects associated with aging. Recent studies in our laboratory demonstrated that the colonic mucosa of elder rats is more susceptible to folate depletion than that of young rats. Depletion of folate results in a shift in the forms of folate in the colon as well as increased uracil incorporation into DNA, a purported mechanism for colonic carcinogenesis. However, modest folate supplementation eliminates evidence of inadequate folate status in the colons of elder rats, suggesting that the relation between age and folate status in the colon might be one mechanism by which aging modulates colorectal cancer risk. Interactions between folate and aging also affect a spectrum of epigenetic and genetic phenomena such as uracil misincorporation, DNA methylation, protein methylation, mitochondrial deletion, and critical gene expression, which could be related to carcinogenesis. Aging and inadequate dietary folate may interact and collectively induce derangements in folate metabolism, thereby provoking subsequent molecular aberrations, which may enhance carcinogenesis. However, folate supplementation appears to reverse these adverse effects of aging, which is potentially of substantial import because the latter is an unmodifiable risk factor.

Ageing, Chronic Alcohol Consumption and Folate are Determinants of Genomic DNA Methylation, P16 Promoter Methylation and the Expression Of P16 in the Mouse Colon

Older age, dietary folate and chronic alcohol consumption are important risk factors for the development of colon cancer. The present study examined the effects of ageing, folate and alcohol on genomic and p16-specific DNA methylation, and p16 expression in the murine colon. Old (aged 18 months; n 70) and young (aged 4 months; n 70) male C57BL/6 mice were pair-fed either a Lieber-DeCarli liquid diet with alcohol (18 % of energy), a Lieber-DeCarli diet with alcohol (18 %) and reduced folate (0.25 mg folate/l) or an isoenergetic control diet (0.5 mg folate/l) for 5 or 10 weeks. Genomic DNA methylation, p16 promoter methylation and p16 gene expression were analysed by liquid chromatography-MS, methylation-specific PCR and real-time RT-PCR, respectively. Genomic DNA methylation was lower in the colon of old mice compared with young mice (P < 0.02) at 10 weeks. Alcohol consumption did not alter genomic DNA methylation in the old mouse colon, whereas it tended to decrease genomic DNA methylation in young mice (P = 0.08). p16 Promoter methylation and expression were higher in the old mouse colon compared with the corresponding young groups. There was a positive correlation between p16 promoter methylation and p16 expression in the old mouse colon (P < 0.02). In young mice the combination of alcohol and reduced dietary folate led to significantly decreased p16 expression compared with the control group (P < 0.02). In conclusion, ageing and chronic alcohol consumption alter genomic DNA methylation, p16 promoter methylation and p16 gene expression in the mouse colon, and dietary folate availability can further modify the relationship with alcohol in the young mouse.

Oestrogen replacement therapy reduces total plasma homocysteine and enhances genomic DNA methylation in postmenopausal women

Although oestrogen replacement therapy (ERT), which can affect the risk of major cancers, has been known to reduce total plasma homocysteine concentrations in postmenopausal women, the mechanisms and subsequent molecular changes have not yet been defined. To investigate the effect of ERT on homocysteine metabolism, thirteen healthy postmenopausal women were enrolled in a double-blind, placebo-controlled, randomized, cross-over study consisting of two 8-week long phases, placebo and conjugated equine oestrogen (CEE; 0.625 mg/d). Concentrations of total plasma homocysteine, vitamin B6 and serum folate and vitamin B12 were measured by conventional methods. Genomic DNA methylation was measured by a new liquid chromatography/MS method and promoter methylation status of the oestrogen receptor (ER)alpha, ERbeta and p16 genes was analysed by methylation-specific PCR after bisulfite treatment. The CEE phase demonstrated a significantly decreased mean of total plasma homocysteine concentrations compared with the placebo phase (8.08 micromol/l (6.82-9.39) v. 9.29 (7.53-11.35), P < 0.05) but there was no difference in the blood concentrations of the three B vitamins. The CEE phase also showed a significantly increased genomic DNA methylation in peripheral mononuclear cells compared with the placebo phase (2.85 (SD 0.12) ng methylcytosine/microg DNA v. 2.40 +/- (SD 0.15) P < 0.05). However, there was no difference in promoter methylation in the ERalpha, ERbeta and p16 genes. This study demonstrates that decreased homocysteinaemia by CEE therapy parallels with increased genomic DNA methylation, suggesting a potential new candidate mechanism by which ERT affects the risk of cancers and a possible new candidate biomarker for the oestrogen-related carcinogenesis through folate-related one-carbon metabolism.

Dose-dependent Effects of Dietary Folate on Aortic Relaxation and Hepatic C-reactive protein Levels in C57BL/6 Mice

Endothelial dysfunction is an initial step in atherosclerosis. B vitamins (B6, B12, and folate) are important contributing factors to vascular homeostasis. Deficiencies in these B vitamins induce cardiovascular diseases by altering vascular homeostasis. Folate plays important roles in nitric oxide homeostasis in the endothelium. To determine the dose-dependent effect of dietary folate on atherosclerosis, we studied aortic relaxation and hepatic C-reactive protein (CRP) levels in C57BL/6 mice. In this study, a total of 54 male C57BL/6, 8-wk old mice were split into 2 dietary groups (control and Western style diet). Each diet group was divided into 3 subgroups according to dietary folate dosage (0.2, 2, and 8 mg/kg). After 18 months, the relaxation response seen in aortic rings from mice fed 0.2 or 2 mg folate/kg in both diet groups. However, the aortic relaxation response was not seen and no differences were observed in mice fed 8mg folate/kg in either diet group (p<0.05). Hepatic CRP levels at all folate dosages (0.2, 2, 8 mg folate/kg) were higher in the groups fed a Western style diet than in mice fed a control diet (p=0.035). CRP levels were lower in mice fed 0.2 mg folate/kg than in mice fed 2 or 8 mg folate/kg in both diet groups (p<0.05). These results indicate that in C57BL/6 mice 0.2 mg folate/kg may be enough to prevent atherosclerosis by inducing the relaxation responses of the aorta and by reducing levels of hepatic CRP, regardless of dietary style.

Abstract A20: Deacetylation of histone H3 at lysine 9 with ethanol in human colonic epithelial cells

A20 Epidemiologic observations have implicated chronic excess ethanol consumption as a risk factor for various cancers including colon cancer. Recent epigenetic studies demonstrated that ethanol causes selective acetylation of histone H3 at lysine 9 (H3K9) in the liver, lung, spleen and testes, indicating that ethanol may inhibit histone deacetylase activity, or enhance histone acetyltransferase activity in those tissues. We therefore investigated the effect of ethanol on histone H3 modifications in colonic epithelial cells. NCM460, human colonic epithelial cells were incubated with 100mM of ethanol for 24, 48, or 72 hr and then the acetylation and methylation states of histone H3K9 were measured by immunoblot analysis using site-specific antibodies. We found that ethanol decreased acetylation of histone H3K9 in a time-dependent manner (71, 21, 17%, respectively, P Citation Information: Cancer Prev Res 2008;1(7 Suppl):A20.